Oxidative modification of macromolecules has been implicated in a large number of physiologic and pathologic conditions. Endogenous sources of oxidants, which result from the incomplete reduction of oxygen to water, include activated white cells, flavoproteins, and reactive oxygen species. Proteins are susceptible to oxidation and are converted to numerous products. Virtually all amino acids have some susceptibility to oxidation, but histidine appears to be particularly reactive under oxidative conditions. Recent studies by Uchida and Kawakishi demonstrate that 2-oxo- histidine might serve as a marker for oxidatively modified proteins. The formation of 2-oxo-histidine in both synthetic and biological systems is of interest in our study of oxidative damage of proteins. Ongoing research combines the techniques of biochemistry, organic chemistry, and cell culture to investigate the products of these reactions. Synthesis of N- benzoyl-2-oxo-histidine using a metal-catalyzed oxidation system consisted of copper, ascorbate, oxygen, and N-benzoylhistidine (a modified method of Uchida and Kawakishi). The product was hydrolyzed to 2-oxo-histidine in the presence of high levels of reducing agents. With the availability of a synthetic sample, studies were conducted to determine the chemical properties of 2-oxo-histidine and to develop an analytical technique to be used with biological systems. The compound is inherently unstable in aqueous solution, while the precursor N-benzoyl-2-oxo-histidine is stable. Using a number of approaches to stabilize the residue, it was shown that three standard chromatographic techniques can now be used to analyze biological samples for the oxidized amino acid. The use of 2-oxo- histidine provides greater specificity than an assay for protein associated carbonyl groups, which is commonly utilized to assess protein oxidation. Using a high performance liquid chromatography method, the formation of 2-oxo-histidine has been correlated with the loss of enzyme activity in oxidized glutamine synthetase. In addition, enolase, trypsin, Cu,Zn-superoxide dismutase, and low density lipoprotein have been shown to contain 2-oxo histidine after exposure to oxidizing conditions. The availability of sensitive biochemical assays for products of protein oxidation will facilitate the study of oxidative stress.